Cerebellum (2008) 7:563–566 DOI 10.1007/s12311-008-0068-2
Simple and Complex Spike Firing Patterns in Purkinje Cells During Classical Conditioning Anders Rasmussen & Dan-Anders Jirenhed & Germund Hesslow
Published online: 18 October 2008 # Springer Science + Business Media, LLC 2008
Abstract Classical blink conditioning is known to depend critically on the cerebellum and the relevant circuitry is gradually being unravelled. Several lines of evidence support the theory that the conditioned stimulus is transmitted by mossy fibers to the cerebellar cortex whereas the unconditioned stimulus is transmitted by climbing fibers. This view has been dramatically confirmed by recent Purkinje cell recordings during training with a classical conditioning paradigm. We have tracked the activity of single Purkinje cells with microelectrodes for several hours in decerebrate ferrets during learning, extinction, and relearning. Paired peripheral forelimb and periocular stimulation, as well as paired direct stimulation of cerebellar afferent pathways (mossy and climbing fibers) causes acquisition of a pause response in Purkinje cell simple spike firing. This conditioned Purkinje cell response has temporal properties that match those of the behavioral response. Its latency varies with the interstimulus interval and it responds to manipulations of the conditioned stimulus in the same way that the blink does. Complex spike firing largely mirrors the simple spike behavior. We have previously suggested that cerebellar learning is subject to a negative feedback control via the inhibitory nucleoolivary pathway. As the Purkinje cell learns to respond to the conditioned stimulus with a suppression of simple spikes, disinhibition of anterior interpositus neurons would be expected to cause inhibition of the inferior olive. Observations of complex spike firing in the Purkinje cells during conditioning and extinction confirm this prediction. A. Rasmussen : D.-A. Jirenhed : G. Hesslow (*) Department of Experimental Medical Science, Lund University, BMC F10, SE 22184 Lund, Sweden e-mail:
[email protected]
Before training, complex spikes are unaffected or facilitated by the conditioned stimulus, but as the simple spike pause response develops, spontaneous and stimulus-evoked complex spikes are also strongly suppressed by the conditioned stimulus. After extinction of the simple spike pause response, the complex spikes reappear. Keywords Simple spike firing . Complex spike firing . Purkinje cells . Classical conditioning . Cerebellum
Introduction: Proposed Role of Climbing Fibers in Classical Conditioning The classical conditioning paradigm, first introduced and explored by Pavlov a century ago, has been applied to autonomic, emotional, and skeletal muscle responses, the latter usually focused on blink conditioning. When it was shown in the early 1980s that the cerebellum was critical for this form of learning [1–3], it was natural to speculate that the conditioned stimulus (CS) signal, often a tone or a light, was carried to the cerebellar cortex by the mossy fibers whereas the unconditioned stimulus (US) signal, usually an air puff to the cornea or electrical stimulation of the periocular area, was carried by climbing fibers from the inferior olive. According to this cerebellar cortical conditioning (CCC) model, this would lead to a decreased firing in Purkinje cells in response the CS [4–6]. This had been explicitly suggested by Albus [7] and received early support in the finding that both mossy and climbing fibers projected to an area known to be related to facial movements [8]. There has been a large amount of evidence supporting the proposed role of the mossy fibers [4], but evidence for the role of the climbing fibers in transmitting the US has
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Recently, however, recordings from single Purkinje cells during conditioning in decerebrate ferrets have provided very strong evidence that the US is carried by the climbing fibers. When ferrets are trained using 300 ms 50 Hz electrical stimulation of forelimb skin as the CS and periorbital stimulation as the US (cf. Fig. 1), Purkinje cells in a blink-controlling area in the c3 zone of the cerebellar cortex [13, 14] change their responsiveness to the CS [15].
Initially, there is either no response at all or a slight increase in simple spike firing during the CS, but after a couple of hours of training (intertrial interval 15–20 s), the Purkinje cells begin to respond with a decreased firing, eventually resulting in a well-timed pause. A typical example is shown in Fig. 2a–b. Just as the behavioral CR, this conditioned Purkinje cell response (“Purkinje cell CR”) is delayed so that the maximum simple spike suppression occurs toward the end of the CS–US period. If the CS–US interval is increased, which normally causes a corresponding further delay in the behavioral CR, the Purkinje cell CR behaves in the same way [15]. It also closely parallels the behavioral CR in several other respects. For instance, it extinguishes slowly with unpaired stimulation and is very rapidly reacquired, often within about ten trials, when paired CS– US stimulation is resumed. In perfect agreement with the CCC model, exactly the same change in Purkinje cell responsiveness occurred when the CS was a direct train of stimuli to the mossy fibers. More importantly in the present context was that Purkinje cell CRs were also acquired when the US consisted of direct electrical stimulation of either the inferior olive or of the ascending climbing fibers in the inferior cerebellar peduncle rather than peripheral stimulation. Thus, three different ways of activating the climbing fibers can support conditioning.
Fig. 1. Experimental setup. a Wiring diagram and experimental setup showing the recording site and the different stimulation sites. PC Purkinje cell, pf parallel fiber, AIN anterior interpositus nucleus, cf climbing fiber, GrC granule cell, IC inferior colliculus. b Sample eyelid electromyogram record of a typical CR on a CS-alone trial. The onset latency is about 100 ms and the duration is about 350 ms. c
Cerebellar cortex of the ferret. The blink-controlling area from which Purkinje cells were sampled is indicated in black. d Sample record from a single Purkinje cell in the area outlined in c with simple spikes and two characteristic complex spikes elicited by electrical stimulation of the periocular skin. Shock artifact indicated by arrow and the complex spikes by asterisks
proven much more elusive. The most natural tests, inactivating the olive or replacing the US with direct olivary stimulation, have serious flaws. Inactivation of the olive with drugs or lesions would, because of the ensuing loss of background climbing fiber input to the Purkinje cells, result in a virtual shutdown of the cerebellum [9–11] and is, therefore, not a specific test of its role in learning. There are reports that direct stimulation of the inferior olive can function as a US [12]. Even when successful, it is difficult to interpret such experiments. Stimulation of the appropriate part of the olive will normally antidromically activate various olivary afferents such as those coming from the trigeminal nucleus, which also provides mossy fiber input to the cerebellum.
Purkinje Cell Simple Spike Firing
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Fig. 2. Simple and complex spike firing in conditioned Purkinje cells. a Acquisition of Purkinje cell CR. Sample records from a Purkinje cell at the start of training (upper record) with paired mossy fiber (CS) and climbing fiber (US) stimulation and after about 600 trials (lower record) when the Purkinje cell CR is fully developed. Bars under the lower record show the timing of CS and US. Calibration bars to the right, 0.5 mV. b Time histograms show the firing pattern of the cell during the first 40 trials (upper panels) and the last 40 trials (lower panel) after about 600 trials of training. Bin width, 10 ms. c Inhibitory
response in inferior olive. The upper panel shows two superimposed histograms of simple spikes (thin gray bars) and complex spikes (wide black bars) on CS-alone trials before training in a Purkinje cell. The firing frequencies are shown as the percentage of background frequencies before CS onset. Bin width, 30 ms. The lower panel shows the firing frequencies in the same cell on CS-alone trials after an inhibitory response in simple spikes has been acquired. Notice the decrease in complex spikes succeeding the Purkinje cell CR in simple spikes
Purkinje Cell Complex Spike Firing
Furthermore, we would expect the Purkinje cell CR to be accompanied by a depression of the inferior olive, observable as a reduced frequency of complex spikes. This expectation was consistently confirmed. In some Purkinje cells, there was a slight asynchronous increase in complex spike frequency during the first half of the CS period, in others there was no change, but in all Purkinje cells (at present, we have data from nine Purkinje cells) with a clear simple spike suppression, there was a marked suppression of complex spike firing following the simple spike pause and just preceding the US. A typical example is shown in Fig. 2c. This clearly confirms the feedback hypothesis. Such a feedback control mechanism could explain some famous paradoxical findings in learning research. For instance, when a subject has learned to respond to one CS, say a light, learning to a second CS, say a tone, presented together with the first one is blocked [18, 19]. Since the first CS would be expected to generate a Purkinje cell CR that disinhibits the cerebellar nucleus and thus
Recordings were also made in these experiments of complex spikes. (Since the complex spike data were obtained in the same experiments, the methodological details are identical to those described in [15].) One reason for studying complex spikes during conditioning was to test the hypothesis proposed previously, that the inhibitory projection from the cerebellar nuclei to the inferior olive functions as a negative feedback system for controlling both the background activity in the Purkinje cells and the amplitude of the Purkinje cell CR [9, 16]. The idea is that, when a full-blown Purkinje CR has been acquired and the Purkinje cell responds to the CS with a suppression of simple spikes, disinhibition of anterior interpositus neurons would be expected to cause inhibition of the inferior olive. This would block the US pathway and prevent further learning. Indeed, stimulation of the nucleo-olivary pathway just before the US in paired trials, which blocks the proposed US pathway, causes extinction of the CR [17].
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inhibits the olive, such blocking is nicely explained by the nucleo-olivary circuit [9, 16]. Cerebellar-induced inhibition of the olive might also explain why, when a subject has learned to respond reliably to two independent CSs, presentation of these CSs together, even when paired with the US, causes extinction, the so-called overexpectation [20, 21]. Although consistent with the available evidence, these suggestions remain to be rigorously tested. Acknowledgements This work was supported by the Swedish Research Council (no. 09899) and the Segerfalk, Söderberg and Åhlen foundations.
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